The gene encoding .beta.-glucuronidase has gained widespread use as a versatile tool for use in a variety of recombinant DNA techniques. The most widely embraced utility of .beta.-glucuronidase is as a reporter gene in .beta.-glucuronidase-deficient cells for indicating patterns of gene expression mediated by sequence elements attached to the glucuronidase gene. In addition, it has been recognized that .beta.-glucuronidase can play a role as a positive selection marker for cells carrying exogenous DNA containing an expressible glucuronidase gene. The utility of .beta.-glucuronidase as selective marker relies on the fact that cells cannot grow on a .beta.-glucuronide carbon source such as a glucuronide disaccharide unless .beta.-glucuronidase is provided to cleave the .beta.-glucuronide bond. The most useful example of such a disaccharide is cellobiuronic acid, which comprises .beta.-glucuronic acid in [1-4] linkage to glucose. Only cells expressing .beta.-glucuronidase can grow on a carbon source consisting only of cellobiuronic acid.
Unlike the toxic agents commonly used in conjunction with negative selection markers in recombinant DNA techniques, cellobiuronic acid is non-toxic. Although methods related to the use of .beta.-glucuronidase are well known, including methods for introducing .beta.-glucuronidase genes into cells, and for assaying .beta.-glucuronidase activity, see, e.g., U.S. Pat. No. 5,599,670 to Jefferson, there are unfortunately few economical methods for preparing cellobiuronic acid.
One known method of preparing cellobiuronic acid is by exposing cellulose to nitrous oxide. The exposure to nitrous oxide results in random oxidation of a portion of the glucose residues to glucuronic acid residues. Subsequent acid hydrolysis can be used to produce cellobiuronic acid, which may be purified from the reaction mixture. This method of preparing cellobiuronic acid is deficient for several reasons. The oxidation step involves the expense and difficulties inherent in performing a controlled oxidation in the presence of a toxic gas. Also, the physical and chemical properties of cellulose usually necessitate a pre-treatment step such as grinding, milling or steam explosion in order to allow optimal accessibility of the acid to the fibrous material during acid hydrolysis. In addition, because the oxidation step is not readily controllable, the cellobiuronic acid produced after oxidation and then hydrolysis is part of a hydrolysate mixture that may contain several byproducts such as oligo-glucuronic acids of various lengths, glucuronic acid, and some gluconic acids. These negatively charged byproducts have chemical properties similar to cellobiuronic acid, which makes purification of cellobiuronic acid by simple procedures such as anion-exchange chromatography or crystallization more difficult and expensive than if cellobiuronic acid were the only negatively charged reaction product.
The preparation of cellobiuronic acid from glucose via a synthetic oxidation approach is thus deficient for several reasons. Therefore, there is a need in the art to provide a simple method for the rapid and economical preparation of cellobiuronic acid. The present invention satisfies this need and provides other related advantages as disclosed further herein.